Potent in vivo anti-tumor activity of isolated CD62L(low) lymph node cells sensitized in vivo with tumor lysate-pulsed DC-based vaccines

BACKGROUND: DC are potent APC that can activate both CD4 and CD8 T cells in vitro and in vivo. Although the efficacy of DC-based cancer vaccines is currently being evaluated in clinical trials, the systemic immune suppression in cancer patients negatively impacts the clinical benefit of this therapeutic approach. Therefore, in this study we tested the feasibility and anti-tumor effect of adoptive immunotherapy using in vitro-activated CD62L(low) lymph node cells that were isolated from DC-vaccinated draining lymph nodes (VDLN). METHODS: DC were prepared from BM cells and loaded with tumor lysate for inoculating into naive mice. Subsequently, the VDLN were removed and CD62L(low) cells in the VDLN population isolated, expanded in vitro by 5-day culture with IL-2 and immobilized anti-CD3 stimulation, then injected into mice with established pulmonary tumors. Eighteen days after treatment, mice were killed in order to enumerate pulmonary tumor nodes. RESULTS: DC phagocytosed the tumor lysate efficiently and induced detectable T-cell responses and significant cell expansion in the draining lymph nodes. After induction of maturation by LPS treatment, DC expressed higher levels of CD40, CD86 and MHC class II molecules. When CD62L(low) VDLN cells that had been isolated and expanded in vitro were transferred into tumor-bearing mice, as few as 3 x 10(6) cells were able to cure metastatic pulmonary tumors in vivo. DISCUSSION: DC-based VDLN T cells are an important source of anti-tumor effector for adoptive immunotherapy. This study provides a novel and an effective protocol using T-cell adoptive immunotherapy for application in cancer patients; therefore, clinical trials based on this protocol may be warranted.     

Behavior of metastatic and nonmetastatic breast tumors in old mice

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.     

Reinforcement of cancer immunotherapy by adoptive transfer of cblb-deficient CD8+ T cells combined with a DC vaccine

The success of cancer immunotherapy is limited by potent endogenous immune-evasion mechanisms, which are at least in part mediated by transforming growth factor-β (TGF-β). The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is established to regulate TGF-β sensitivity. cblb-deficient animals reject tumors via CD8(+) T cells, which make Cbl-b an ideal target for improvement of adoptive T-cell transfer (ATC) therapy. In this study, we show that cblb-deficient CD8(+) T cells are hyper-responsive to T-cell receptor (TCR)/CD28-stimulation and are in part protected against the negative cues induced by TGF-β in vitro. Notably, adoptive transfer of polyclonal, non-TCR transgenic cblb-deficient CD8(+) T cells is not sufficient to reject B16-ova or EG7 tumors in vivo. Thus, cblb-deficient ATC requires proper in vivo re-activation by a dendritic cell (DC) vaccine. In strict contrast to ATC monotherapy, this approach delayed tumor outgrowth and significantly increased survival rates, which is paralleled by increased CD8(+) T-cells infiltration to the tumor site and enrichment of ova-specific and interferon-γ (IFN-γ)-secreting CD8(+) T cell in the draining lymph node (LN). Moreover, CD8(+) T cells from cblb-deficient mice vaccinated with the DC vaccine show increased cytolytic activity in vivo. In summary, our data using cblb-deficient polyclonal, non-TCR-transgenic adoptively transferred CD8(+) T cells into immuno-competent non-lymphodepleted recipients suggest that targeting Cbl-b might serve as a novel 'adjuvant approach', suitable to augment the effectiveness of established anti-cancer immunotherapies.     

Adjuvant immunotherapy of experimental autoimmune encephalomyelitis: immature myeloid cells expressing CXCL10 and CXCL16 attract CXCR3+CXCR6+ and myelin-specific T cells to the draining lymph nodes rather than the central nervous system

CFA is a strong adjuvant capable of stimulating cellular immune responses. Paradoxically, adjuvant immunotherapy by prior exposure to CFA or live mycobacteria suppresses the severity of experimental autoimmune encephalomyelitis (EAE) and spontaneous diabetes in rodents. In this study, we investigated immune responses during adjuvant immunotherapy of EAE. Induction of EAE in CFA-pretreated mice resulted in a rapid influx into the draining lymph nodes (dLNs) of large numbers of CD11b(+)Gr-1(+) myeloid cells, consisting of immature cells with ring-shaped nuclei, macrophages, and neutrophils. Concurrently, a population of mycobacteria-specific IFN-γ-producing T cells appeared in the dLNs. Immature myeloid cells in dLNs expressed the chemokines CXCL10 and CXCL16 in an IFN-γ-dependent manner. Subsequently, CD4(+) T cells coexpressing the cognate chemokine receptors CXCR3 and CXCR6 and myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+) T cells accumulated within the chemokine-expressing dLNs, rather than within the CNS. Migration of CD4(+) T cells toward dLN cells was abolished by depleting the CD11b(+) cells and was also mediated by the CD11b(+) cells alone. In addition to altering the distribution of MOG-specific T cells, adjuvant treatment suppressed development of MOG-specific IL-17. Thus, adjuvant immunotherapy of EAE requires IFN-γ, which suppresses development of the Th17 response, and diverts autoreactive T cells away from the CNS toward immature myeloid cells expressing CXCL10 and CXCL16 in the lymph nodes.     

The role of adoptively transferred CD8 T cells and host cells in the control of the growth of the EG7 thymoma: factors that determine the relative effectiveness and homing properties of Tc1 and Tc2 effectors

We had previously examined the factors that regulate the response of OVA-specific TCR-transgenic CD8 T cells to the B16 OVA melanoma, growing as lung metastases. We examine here whether the same parameters operate for EG7, growing intradermally. Tc1 or Tc2 CD8 effector cells from OT-1 mice were injected either mixed with the tumor or i.v. at day 0 or 7. Tc2 were one-fifth to one-tenth as effective as Tc1 when injected with the tumor, in controlling tumor growth, but were only 1/20 to 1/100 injected i.v. Tc1 injected i.v. entered the draining lymph nodes faster than Tc2 and caused a faster accumulation of host cells. Both caused an abrupt termination of host cell entry into lymph nodes and spleen after tumor elimination, but this occurred earlier for Tc1 than for Tc2. Host responses were ineffective in the absence of adoptive transfer but were essential after transfer. Perforin expression in the donor cells plays no role in adoptively transferred Tc1 or Tc2 control of the tumor, and neither IL-4 nor IL5 is needed for Tc1 or Tc2 function. Tc1 cells from mice lacking IFN-gamma, however, control tumor growth less well, whereas Tc2 effectors lacking IFN-gamma are unaffected. Tc1 from IFN-gamma-deficient mice attract fewer host cells to the draining lymph node, whereas Tc1 cells from perforin-deficient donors are unimpaired. We conclude that host cell recruitment is a crucial element in adoptive immunotherapy. The differences between the EG7 and the previous B16 melanoma model are discussed.     

Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.     

Activation, differentiation, and migration of naive virus-specific CD8+ T cells during pulmonary influenza virus infection

The low precursor frequency of individual virus-specific CD8(+) T cells in a naive host makes the early events of CD8(+) T cell activation, proliferation, and differentiation in response to viral infection a challenge to identify. We have therefore examined the response of naive CD8(+) T cells to pulmonary influenza virus infection with a murine adoptive transfer model using hemagglutinin-specific TCR transgenic CD8(+) T cells. Initial activation of CD8(+) T cells occurs during the first 3 days postinfection exclusively within the draining lymph nodes. Acquisition of CTL effector functions, including effector cytokine and granule-associated protease expression, occurs in the draining lymph nodes and differentially correlates with cell division. Division of activated CD8(+) T cells within the draining lymph nodes occurs in an asynchronous manner between days 3 and 4 postinfection. Despite the presence of Ag for several days within the draining lymph nodes, dividing T cells do not appear to maintain contact with residual Ag. After multiple cell divisions, CD8(+) T cells exit the draining lymph nodes and migrate to the infected lung. Activated CD8(+) T cells also disseminate throughout lymphoid tissue including the spleen and distal lymph nodes following their emigration from draining lymph nodes. These results demonstrate an important role for draining lymph nodes in orchestrating T cell responses during a local infection of a discrete organ to generate effector CD8(+) T cells capable of responding to infection and seeding peripheral lymphoid tissues.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Effects of phthalate esters on dendritic cell subsets and interleukin-4 production in fluorescein isothiocyanate-induced contact hypersensitivity

Phthalate esters with short alkyl chains, such as di-ethyl (DEP), di-n-propyl (DPP), and di-butyl phthalate (DBP), have adjuvant effects on an FITC-induced contact hypersensitivity mouse model. The adjuvant effects of DPP and DBP are associated with enhanced trafficking of FITC-presenting CD11b(+) dendritic cells (DC). DEP has relatively weak activity as to FITC-positive cell migration. Here we demonstrated that DBP and DPP also increased the number of FITC-positive CD8alpha(+) DC in draining lymph nodes. We also found enhanced production of interleukin-4 in draining lymph nodes after FITC sensitization with DEP, DPP, or DBP, suggesting an additional adjuvant mechanism of phthalate esters.     

Mechanical Disruption of Tumors by Iron Particles and Magnetic Field Application Results in Increased Anti-Tumor Immune Responses

The primary tumor represents a potential source of antigens for priming immune responses for disseminated disease. Current means of debulking tumors involves the use of cytoreductive conditioning that impairs immune cells or removal by surgery. We hypothesized that activation of the immune system could occur through the localized release of tumor antigens and induction of tumor death due to physical disruption of tumor architecture and destruction of the primary tumor in situ. This was accomplished by intratumor injection of magneto-rheological fluid (MRF) consisting of iron microparticles, in Balb/c mice bearing orthotopic 4T1 breast cancer, followed by local application of a magnetic field resulting in immediate coalescence of the particles, tumor cell death, slower growth of primary tumors as well as decreased tumor progression in distant sites and metastatic spread. This treatment was associated with increased activation of DCs in the draining lymph nodes and recruitment of both DCs and CD8(+)T cells to the tumor. The particles remained within the tumor and no toxicities were observed. The immune induction observed was significantly greater compared to cryoablation. Further anti-tumor effects were observed when MRF/magnet therapy was combined with systemic low dose immunotherapy. Thus, mechanical disruption of the primary tumor with MRF/magnetic field application represents a novel means to induce systemic immune activation in cancer.     

Phenotype and homing of CD4 tumor-specific T cells is modulated by tumor bulk

Technical difficulties in tracking endogenous CD4 T lymphocytes have limited the characterization of tumor-specific CD4 T cell responses. Using fluorescent MHC class II/peptide multimers, we defined the fate of endogenous Leishmania receptor for activated C kinase (LACK)-specific CD4 T cells in mice bearing LACK-expressing TS/A tumors. LACK-specific CD44(high)CD62L(low) CD4 T cells accumulated in the draining lymph nodes and had characteristics of effector cells, secreting IL-2 and IFN-gamma upon Ag restimulation. Increased frequencies of CD44(high)CD62L(low) LACK-experienced cells were also detected in the spleen, lung, liver, and tumor itself, but not in nondraining lymph nodes, where the cells maintained a naive phenotype. The absence of systemic redistribution of LACK-specific memory T cells correlated with the presence of tumor. Indeed, LACK-specific CD4 T cells with central memory features (IL-2(+)IFN-gamma(-)CD44(high)CD62L(high) cells) accumulated in all peripheral lymph nodes of mice immunized with LACK-pulsed dendritic cells and after tumor resection. Together, our data demonstrate that although tumor-specific CD4 effector T cells producing IFN-gamma are continuously generated in the presence of tumor, central memory CD4 T cells accumulate only after tumor resection. Thus, the continuous stimulation of tumor-specific CD4 T cells in tumor-bearing mice appears to hinder the systemic accumulation of central memory CD4 T lymphocytes.     

Intralesional injection of adenovirus encoding CC chemokine ligand 16 inhibits mammary tumor growth and prevents metastatic-induced death after surgical removal of the treated primary tumor

The CC chemokine ligand (CCL)16 exerts chemotactic activity on human monocytes and lymphocytes. Although no murine homologous has been defined, the TSA mouse adenocarcinoma cells engineered to express human CCL16 are rapidly rejected by syngenic mice. An adenovirus encoding CCL16 (AdCCL16) was generated using a Cre-Lox-based system and was used to determine whether this chemokine might also block pre-existing tumors. Both recombinant and viral CCL16 showed in vitro chemotactic activity for murine CD4(+) and CD8(+) lymphocytes and dendritic cells (DC). AdCCL16, but not the control empty vector, when injected in established nodules significantly delayed tumor growth. Immunohistochemistry revealed accumulation of CD4(+) and CD8(+) T cells and DC in the treated tumors as well as in draining lymph nodes. DC from such lymph nodes stimulated IFN-gamma by a T cell clone specific for the known TSA tumor-associated Ag (TAA), suggesting the tumor origin of these cells. Lymphocytes from the same nodes showed specific CTL activity against TSA tumor cells and their immunodominant TAA peptide. Antitumor activity required CD4, CD8, and IFN-gamma production, as shown using subset-depleted and knockout mice. Despite the robust and rapid immune response triggered by intratumoral injection of AdCCL16, the lesions were not completely rejected; however, the same treatment given before surgical excision of primary lesions prevented metastatic spread and cured 63% of mice bearing the 4T1 mammary adenocarcinoma, which is perhaps the most compelling model of spontaneous metastasis.     

CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells

CD4(+) T cells control the effector function, memory, and maintenance of CD8(+) T cells. Paradoxically, we found that absence of CD4(+) T cells enhanced adoptive immunotherapy of cancer when using CD8(+) T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4(+)CD25(-) Th cells (Th cells) with tumor/self-reactive CD8(+) T cells and vaccination into CD4(+) T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4(+) T cells that contained a mixture of Th and CD4(+)CD25(+) T regulatory cells (T(reg) cells) or T(reg) cells alone prevented effective adoptive immunotherapy. Maintenance of CD8(+) T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2(-/-) mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring T(reg) cells to be effective.     

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.     

Augmentation versus inhibition: effects of conjunctional OX-40 receptor monoclonal antibody and IL-2 treatment on adoptive immunotherapy of advanced tumor.

Therapeutic efficacy of adoptive immunotherapy of malignancies is proportional to the number of effector T cells transferred. Traditionally, exogenous IL-2 treatment has been used to promote the survival and function of transferred cells. Recently, we described the therapeutic effects of in vivo ligation of the costimulatory receptor, OX-40R, on activated T cells during early tumor growth. In this study, we examined the effects of IL-2 and OX-40R mAb on adoptive immunotherapy of advanced tumors. For treatment of 10-day 3-methylcholanthrene 205 pulmonary metastases, systemic transfer of 50 x 10(6) activated tumor-draining lymph node T cells resulted in >99% reduction of metastatic nodules. With either IL-2 or OX-40R mAb conjunctional treatment, only 20 x 10(6) cells were required. Advanced 10-day 3-methylcholanthrene 205 intracranial tumors could be cured by the transfer of 15 x 10(6) L-selectin(low) T cells derived from draining lymph nodes. In this situation, IL-2 administration inhibited therapeutic effects of the transferred cells. By contrast, 5 x 10(6) T cells were sufficient to cure all mice if OX-40R mAb was administrated. Studies on trafficking of systemically transferred T cells revealed that IL-2, but not OX-40R mAb, impeded tumor infiltration by T cells. Tumor regression required participation of both CD4 and CD8 T cells. Because only CD4 T cells expressed OX-40R at cell transfer, direct CD4 T cell activation is possible. Alternatively, OX-40R might be up-regulated on transferred T cells at the tumor site, rendering them reactive to the mAb. Our study suggests OX-40R mAb to be a reagent of choice to augment T cell adoptive immunotherapy in clinical trials.     

Systemic IL-1 and adjuvant treatment of an experimental tumor

In this investigation, systemic administration of interleukin-1 (IL-1) and local adjuvant therapy were shown to modify immunological parameters associated with the lymphatics draining the site of experimental tumor inoculation. These immunological parameters were shown to be modified early (within 7 days) following tumor inoculation and within the time period of IL-1 administration. IL-1 induced a marked increase in the number of lymphocytes within the brachial and axillary lymph nodes associated with the tumor inoculation site. This increase was characterized by an overall augmentation in the number of CD8⁺ and CD4⁺ lymphocytes.In vitro, these lymph node cells showed enhanced proliferation in response to interleukin-2 (IL-2) when compared to non-IL-1 treated animals, and were capable of mounting a potentially greater cytotoxic response for both NK sensitive and NK resistant tumor targets. Without IL-1 administration, temporal and sequential lymph node cellular changes were observed, but were diminished and delayed when compared to the IL-1 treated animals. By adoptive transfer of tumor resistance, lymph node cells from IL-1 treated animals were demonstrated to be tumor-protectivein vivo. These results demonstrate that systemic IL-1 induces regional changes in the lymphatics of mice undergoing primary tumor challenge with adjuvant therapy and that these changes result in tumor protection for the host.     

Tumor immunotherapy by epicutaneous immunization requires langerhans cells

A role for Langerhans cells (LC) in the induction of immune responses in the skin has yet to be conclusively demonstrated. We used skin immunization with OVA protein to induce immune responses against OVA-expressing melanoma cells. Mice injected with OVA-specific CD8(+) T cells and immunized with OVA onto barrier-disrupted skin had increased numbers of CD8(+) T cells in the blood that produced IFN-gamma and killed target cells. These mice generated accelerated cytotoxic responses after secondary immunization with OVA. Prophylactic or therapeutic immunization with OVA onto barrier-disrupted skin inhibited the growth of B16.OVA tumors. LC played a critical role in the immunization process because depletion of LC at the time of skin immunization dramatically reduced the tumor-protective effect. The topically applied Ag was presented by skin-derived LC in draining lymph nodes to CD8(+) T cells. Thus, targeting of tumor Ags to LC in vivo is an effective strategy for tumor immunotherapy.     

Mast cell migration from the skin to the draining lymph nodes upon ultraviolet irradiation represents a key step in the induction of immune suppression

The UV radiation in sunlight is the primary cause of skin cancer. UV is also immunosuppressive and numerous studies have shown that UV-induced immune suppression is a major risk factor for skin cancer induction. Previous studies demonstrated that dermal mast cells play a critical role in the induction of immune suppression. Mast cell-deficient mice are resistant to the immunosuppressive effects of UV radiation, and UV-induced immune suppression can be restored by injecting bone marrow-derived mast cells into the skin of mast cell- deficient mice. The exact process however, by which mast cells contribute to immune suppression, is not known. In this study, we show that one of the first steps in the induction of immune suppression is mast cell migration from the skin to the draining lymph nodes. UV exposure, in a dose-dependent manner, causes a significant increase in lymph node mast cell numbers. When GFP(+) skin was grafted onto mast cell-deficient mice, we found that GFP(+) mast cells preferentially migrated into the lymph nodes draining the skin. The mast cells migrated primarily to the B cell areas of the draining nodes. Mast cells express CXCR4(+) and UV exposure up-regulated the expression of its ligand CXCL12 by lymph node B cells. Treating UV-irradiated mice with a CXCR4 antagonist blocked mast cell migration and abrogated UV-induced immune suppression. Our findings indicate that UV-induced mast cell migration to draining lymph nodes, mediated by CXCR4 interacting with CXCL12, represents a key early step in UV-induced immune suppression.